Lung cancer is the fifth most common cancer and a leading cause of cancer mortality in Australia. Lung cancer has a 5-year survival of 24% and treatment options include surgery and chemotherapy with the possible addition of targeted therapies and immunotherapies. However, more than half of all lung cancers have metastases present at diagnosis with a 5-year survival of 8%. There is an urgent need to develop new targeted therapies for lung cancer. A comprehensive list of gene dependencies in lung cancer has come to the fore with the preclinical application of functional genetic screens using the CRISPR Cas9 system. The system interrogates single gene dependencies with limitations in genomically heterogeneous cancers like lung adenocarcinoma (LUAD).
Here, we used the Cas12a system to probe genetic interactions and dependencies to exploit the synthetic lethal interactions. We synthesized novel combinatorial libraries targeting epigenetic regulators (400 genes and 20 controls with 2 gRNA per gene and 89,830 gRNA combinations) and druggable targets (204 genes and 20 controls with 2 gRNA per gene, and 92,728 combinations of gRNA). Cas12 expressing A549 and H358 LUAD cell lines were transduced with pooled chromatin and drug combinatorial knockout libraries at MOI of 0.3 at a minimum representation of 500x. 300 million cells were plated and transduced with pooled lentivirus from each library and a minimum of 50 million cells were maintained at every passage in duplicate. The libraries were selected with 4ug/ml of puromycin over 35 days. DNA was isolated and library prep was performed using block PCR and purified using magnetic beads. Each library was sequenced with a minimum of 100 million reads and enrichment of gRNA was determined relative to plasmid DNA reads. We found a significant number of gene pairs that conferred synthetic lethality in both A549 and H358 across both chromatin and drug libraries. We are further validating gene pairs of interest using independent gRNA and available drugs.